Enabling immunotherapy for high-risk Group 3 medulloblastoma via systems immunology

项目摘要/摘要 这个项目的目标是剖析免疫逃避机制，并为儿童提供免疫治疗。 通过系统免疫学方法治疗高危第3组髓母细胞瘤(G3MB)。脑瘤是 儿童癌症相关死亡的主要原因。摘要髓母细胞瘤是儿童最常见的恶性肿瘤。 脑瘤，主要有四个分子亚群，其中G3MB最具侵袭性 形式和特点MYC过度表达。免疫抑制肿瘤微环境(TME)差 在G3MB中被理解，并且没有免疫疗法可用于患有这种高危疾病的儿童。系统 免疫学方法--特别是单细胞和空间多组学分析和体内CRISPR 功能筛查-已被证明在解剖肿瘤-TME相互作用和识别新的 免疫治疗的靶点是各种癌症类型，但很少有研究整合这些方法。在我们的 初步研究，我们应用了两种独特的具有免疫能力的基因工程小鼠模型(GEMM) 对MYC驱动的G3MB进行scRNA-seq、scATAC-seq和空间转录分析。我们丰富了 免疫细胞从TME中通过分选CD45阳性细胞进行单细胞研究。我们初步分析了 单细胞和空间组学数据显示神经干细胞样瘤细胞与 巨噬细胞和其他免疫细胞可能产生抑制TME并推动免疫逃避 鼠标G3MB。我们还使用GEMM在肿瘤细胞中进行了体内CRISPR筛查，以识别 肿瘤发展的调节因子，这证明了体内功能基因组学筛选的可行性 在我们的临床前模型中。在这个项目中，首先，我们建议利用尖端的单细胞和空间组学 技术来表征两个G3MB GEMM在肿瘤进展的不同阶段。我们将使用我们的 基于网络的工具，整合这些多组学数据，以剖析动态的肿瘤-免疫相互作用和 在G3MB进程中驱动免疫排斥和抑制的潜在“隐藏”驱动因素。我们会 还验证了在患者样本中进行的小鼠研究中发现的G3MB。我们将开发一个基于云的门户 可视化和探索我们的单细胞和空间数据以及G3MB的肿瘤-TME相互作用。第二，我们将 建立肿瘤-T细胞相互作用的机制基础和实现过继T细胞治疗的策略 G3MB通过发现肿瘤细胞和T细胞中的功能驱动因素和可能的靶点。为此，我们将 在免疫活性GEMM中应用候选方法和体内CRISPR筛选来识别肿瘤- 内源性调节剂将重塑抑制性TME，并使G3MB肿瘤对过继T细胞和 CAR-T细胞疗法。我们还将测试针对T细胞功能的靶向抑制因子是否会启用和优化 针对此类肿瘤的有效的过继T细胞疗法。我们的研究承诺提供新的见解 肿瘤-TME在G3MB中的相互作用机制，并显示合法的免疫治疗机会。

PROJECT SUMMARY / ABSTRACT The goal of this project is to dissect the immune evasion mechanisms and enable immunotherapy for children with high-risk Group 3 medulloblastoma (G3MB) via systems immunology approaches. Brain tumors are the leading cause of cancer-related deaths in children. Medulloblastoma is the most prevalent malignant pediatric brain tumor and is characterized by four major molecular subgroups, among which G3MB is the most aggressive form and features MYC overexpression. The immunosuppressive tumor microenvironment (TME) is poorly understood in G3MB, and no immunotherapy is available for children with this high-risk disease. Systems immunology approaches—especially single-cell and spatial multi-omics profiling and in vivo CRISPR-based functional screening—have proven powerful in dissecting tumor–TME interactions and identifying novel immunotherapy targets in various cancer types, but very few studies have integrated these approaches. In our preliminary studies, we applied two unique immunocompetent genetically-engineered mouse models (GEMMs) of MYC-driven G3MB and performed scRNA-seq, scATAC-seq and spatial transcriptomics profiling. We enriched immune cells from the TME by sorting CD45 positive cells for single-cell studies. Our preliminary analysis of single-cell and spatial omics data revealed striking interactions of neural stem cell-like tumor cells with macrophages and other immune cells that potentially create a suppressive TME and drive immune evasion in mouse G3MB. We also performed in vivo CRISPR screening in tumor cells using the GEMMs to identify modulators of tumor development, which demonstrated the feasibility of in vivo functional genomics screening in our preclinical models. In this project, first, we propose to utilize cutting-edge single-cell and spatial omics technologies to characterize the two G3MB GEMMs at different stages of tumor progression. We will use our network-based tools to integrate these multi-omics data to dissect the dynamic tumor–immune interactions and underlying “hidden” drivers that drive the immune exclusion and suppression during G3MB progression. We will also validate discoveries of G3MB from mouse studies in patient samples. We will develop a cloud-based portal to visualize and explore our single-cell and spatial data and tumor–TME interactomes of G3MB. Second, we will establish the mechanistic basis of tumor–T cell interactions and strategies to enable adoptive T cell therapy for G3MB by discovering functional drivers and putative targets in both tumor cells and T cells. To this end, we will apply both candidate approach and in vivo CRISPR screening in immunocompetent GEMMs to identify tumor- intrinsic modulators that will remodel the suppressive TME and sensitize G3MB tumors to adoptive T cell and CAR-T cell therapies. We will also test if targeting inhibitory factors for T cell function will enable and optimize effective adoptive T cell therapies against such tumors. Our studies promise to provide new insights into mechanisms of tumor–TME interactions in G3MB and manifest legitimate immunotherapeutic opportunities.